Effect of fibroblast growth factor signaling on cumulus expansion in mice in vitro.

The expansion of cumulus cells associated with oocytes is an essential phenomenon in normal mammalian ovulation. Indeed, attenuated expression of cumulus expansion-related genes, including Has2, Ptgs2, Ptx3, and Tnfaip6, results in ovulation failure, leading to female subfertility or infertility. Moreover, emerging evidence suggests that proteins of the fibroblast growth factor (FGF) family, produced within ovarian follicles, regulate the development and function of cumulus cells; however, the effects of FGF signaling on cumulus expansion have not been investigated extensively. Herein, we investigate the effects of FGF signaling, particularly those of FGF8 secreted by oocytes, on epidermal growth factor-induced cumulus expansion in mice. The phosphorylation level of MAPK3/1, an intracellular mediator of FGF signaling, was significantly decreased in cumulus-oocyte complexes (COCs) following treatment with NVP-BGJ398, an FGF receptor inhibitor. Moreover, even though NVP-BGJ398 treatment did not affect cumulus cell expansion, it significantly upregulated the expression of Ptgs2 and Ptx3. In contrast, treatment with recombinant FGF8 did not affect the degree of cumulus expansion or the expression of expansion-related genes in COCs or oocytectomized cumulus cell complexes. Collectively, these results suggest that FGFs, other than FGF8, exert suppressive effects on the cumulus expansion process in mice.

Expression and regulation of estrogen receptor 2 and its coregulators in mouse granulosa cells.

The cooperative effects of estrogen and oocyte-derived paracrine factors (ODPFs) play critical roles in the normal development of ovarian follicles; however, the mechanism underlying this cooperation has not been well studied. The present study aimed to determine whether ODPFs affect estrogen signaling by regulating the expression of estrogen receptor (ESR) and its coregulators in mouse granulosa cells. Some transcripts encoding ESR coregulators were differentially expressed between cumulus and mural granulosa cells (MGCs). The transcript levels of ESR coregulators, including nuclear receptor corepressor 1 and activator 2, in cumulus cells were significantly suppressed by ODPFs; however, they increased when cumulus cell-oocyte complexes were treated with the transforming growth factor beta receptor I inhibitor, SB431542. Moreover, MGCs exhibited significantly higher ESR2 protein and transcript levels than those in cumulus cells. ODPFs promoted Esr2 expression in cumulus cells but had no effect on that in MGCs. Overall, regulation of the expression of ESR2 and its coregulators in cumulus cells by oocytes seems to be one of the mechanisms underlying estrogen-oocyte cooperation in well-developed antral follicles in mice.

Generation of genetically modified mice using SpCas9-NG engineered nuclease.

Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a protospacer-adjacent motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5'-NGG sequence, SpCas9-NG recognized the 5'-NGA, 5'-NGC and 5'-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.

Characterization of mRNA profiles of the exosome-like vesicles in porcine follicular fluid.

Extracellular vesicles such as exosomes contain several types of transcripts, including mRNAs and micro RNAs (miRNAs), and have emerged as important mediators of cell-to-cell communication. Exosome-like vesicles were identified in the ovarian follicles of several mammalian species. Although the miRNA contents have been extensively characterized, the detailed investigation of their mRNA profiles is lacking. Here, we characterize the mRNA profiles of exosome-like vesicles in ovarian follicles in a pig model. The mRNA contents of the exosome-like vesicles isolated from porcine follicular fluid were analyzed and compared with those from mural granulosa cells (MGCs) using the Illumina HiSeq platform. Bioinformatics studies suggested that the exosomal mRNAs are enriched in those encoding proteins involved in metabolic, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) -protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) pathways. While the mRNA profile of the exosome-like vesicles resembled that of MGCs, the vesicles contained mRNAs barely detectable in MGCs. Thus, while the majority of the vesicles are likely to be secreted from MGCs, some may originate from other cell types, including theca cells and oocytes, as well as the cells of non-ovarian organs/tissues. Therefore, the mRNA profiles unveiled several novel characteristics of the exosome-like vesicles in ovarian follicles.